1. Overview Mouse embryonic stem cells Human embryonic stem cells Pluripotency genes and network Long-term self-renewal Directed differentiation Induced pluripotent stem cells

2. Stem cells, pluripotency and differentiation Pluripotency The ability to give rise to differentiated cell types derived from all three primary germ layers of the embryo: endoderm, mesoderm, and ectoderm Two major types of stem cells Adult and embryonic stem cells Induced pluripotent stem (iPS) cells Self-renewal The ability to undergo symmetrical divisions without differentiation induction of pluripotent stem cells from differentiated cells

3. Differentiation of human tissues

4. Generation of embryonic stem cells Two prominent features of ESCs: long-term self-renewal and pluripotency

5. Inner cell mass (ICM): a cluster of cells at the blastocyst stage Day 1Day 2Day 3Day 4DAPI A blastocyst cultured on a petri dish Alkaline phosphatase positive

6. Isolation of ICM cells Rabbit Anti-mouse serum Pipetting Outer cells are lysed. Mouse embryos

7. Derivation of embryonic stem cells from mouse embryos Evans, M.J. & Kaufman, M.H. Nature 292, 154-156, 1981 Martin Evans 2007 Nobel Prize Karyotype is normal

8. Feeders provide factors that maintain embryonic stem cell growth Day 13 mouse embryos Remove heads and internal organs Treat with trypsin and plate cells into a dish MEFs MEFs: mouse embryonic fibroblasts irradiated to stop MEF growth

9. Embryonic stem cells are pluripotent Embryoid bodies (mixture of differentiated cells) Cells of three germ layers Low attachment Mouse injection Teratomas ESCs

10. Thomson, et al., Science, 1998 Derivation of embryonic stem cells from human embryos Jamie Thomson Univ. of Wisconsin Critical factors: MEFs, basic FGF ICM-derived H9 cell line Differentiating cells

11. What are the promises? Understand early human development (infertility, birth defects) and control of cell division (cancer) Cell-based therapy Reduce need for organ and tissue donors/transplants Replace mutant or damaged cells for treatment of diseases such as Parkinson’s disease, spinal cord injury, muscular dystrophy, heart disease, liver dysfunction, osteoporosis, vision and hearing loss A short-cut for drug discovery and testing

12. Transcription factors required for pluripotency Oct4 -/- cells are not pluripotent Oct4 -/- embryo lack inner cell mass Inner cell mass Other important transcription factors: Sox2 and Nanog Austin Smith

14. Core ES cell regulatory circuitry Jaenisch and Young, Cell. 2008

15. He S et al. 2009. Annu Rev Cell Dev Biol; Mouse ESCs LIF (Smith et al., Nature, 1988) BMP (or serum) (Ying et al, Cell, 2003) 3i (Ying et al, Nature, 2008) LIF and BMP act on downstream differentiation signals of MAPK (Buehr et al, Cell, 2008) Regulation of long-term self renewal

16. Directed ES cell differentiation Transcription factor landscape Graf T and Enver T, 2009, Nature

17. What would be an ideal method for directed differentiation? Rapid Simple Cheap Mimic development

18. Conditions for directed differentiation 1. EBs 2. Co-culture 3. Monolayer cultures hESCs EB medium 18 d EB formation EB digestion Hematopoietic stem cells OP9 co- culture Expansion Progenitor Expansion medium 7d Hematopoietic stem cells Terminal differentiation medium 6-7 d Neutrophils OP9 mouse stroma cells – hematopoietic differentiation PA6 or MS5 – neural differentiation

19. Hypothesis Differentiated somatic cells can be re- programmed into pluripotent stem (ESC- like) cells with gene(s) important for ESC identity (pluripotency and self-renewal) Shinya Yamanaka Kyoto University These cells would Bypass ethical issues Create patient-specific pluripotent stem cells

20. 24 candidate genes Dppa2  -cateninOct4 Dppa3/ StellaDnmt3lRex1 Dppa4Fthl17Sall4 Dppa5/ Esg1Grb2Utf1 Ecat1Sox2 Ecat3/ Fbx15Sox15Klf4 Ecat5/ ErasTcl1Myc Ecat8Nanog Ecat9/ Gdf3Stat3 Takahashi and Yamanaka (2006) Cell 126, 663-676 Gene delivery: Retrovirus allowing gene integration into the host genome

21. Putting all 24 genes into MEFs “reprograms” Takahashi and Yamanaka (2006) Cell 126, 663-676 FBX15: an ESC-specific gene; only expressed in ESCs Viral promoter  geo: G418 (an antibiotics that kills the cells) resistance gene So, cells can survive only when they become ESC-like cells

22. Takahashi and Yamanaka (2006) Cell 126, 663-676 Narrowing down the candidates Oct4 (14) Sox2 (15) Klf4 (20) Myc (22)

23. Takahashi and Yamanaka (2006) Cell 126, 663-676 iPS cells are pluripotent Pluripotency markers EB formation Teratoma formation - Saw the same thing with tail-tip fibroblasts

24. Takahashi K., et al. (2007) “Induction of pluripotent stem cells from adult human fibroblasts by defined factors.” Cell 131, 861-72. OCT4, SOX2, KLF4, MYC Yu J., et al. (2007) “Induced pluripotent stem cell lines derived from human somatic cells” Science 318, 1917-1920. OCT4, SOX2, NANOG, LIN28 Park I.H., et al. (2007) “Reprogramming of human somatic cells to pluripotency with defined factors” Nature 451, 141-146. OCT4, SOX2, KLF4, MYC How about human cells?

25. Adapted from: Gepstein. Circ Res 2002 & http://stemcells.nih.gov/info/media/DSC_1187.jpg Translation Derivation Regenerative Medicine Stem Cell Biology Differentiation Tissue morphogenesis Cellular therapies Propagation Scale UpScale Up Quantitative, systematicQuantitative, systematic approaches approaches Quality controlQuality control Stem cell-based therapy iPSCs Human somatic cells “Personalized medicine”

26. Pitfalls with iPSCs Low efficiency of derivation <0.1% Use of C-myc Transgene integration Are they really the same as ESCs?

27. - Are all four genes expressed in the same cells? Approach: Using a single retroviral or lentiviral vector instead of four vectors (2A peptide) Somers A, et al 2010, Stem Cells (STEMCCA Cre-Excisable lentivector) Staerk, J et al, 2010, Cell Stem Cell (T cells and myeloid cells) - Chemical complementation (e.g., with small molecules such as VPA) to replace C-Myc Other compounds: Vitamin C, sodium butyrate, ALK5 inhibitor(*, mESC medium), Apigenin and Luteolin (E-cadherin enhancing) Reprogramming with small molecules only? Low efficiency of derivation Use of C-myc

28. Transgene integration - integrating-free vectors Cre/loxP-recombination system to deliver followed by removal with Cre- recombinase Episomal vectors followed by selection of integration free cells, Single-vector reprogramming system combined with a piggyBac transposon

29. Delivery of OCT-4, SOX2, Myc and Klf4 mRNA or proteins, instead of genes, into somatic cells - Protein and mRNA-based Protein: polyarginine tag Mouse, 30 days, the need for VPA. Human, 50 days, HEK293 cell extracts Synthetic mRNA: 17 days, 2% efficiency

30. Are iPSCs as good as ESCs? Can contribute to embryonic development (Takahashi and Yamanaka, Cell, 2006) Produce adult chimera and are germ-line competent (Okita et al, Nature, 2007) Are capable of giving rise to every cell in the new born mice (Zhao et al., Nature, 2009) Mouse iPSCs: Journal of Molecular Cell Biology (2010), 2, 171–172

31. Human iPSCs At least for some clones, iPSCs are similar if not indistinguishable from ESCs (Mikkelsen et al., Nature, 2008) 1.Global gene expression profiling; 2. Modifications of histone tails; 3. The state of X chromosome inactivation 4. Profiles of DNA methylation

32. Adapted from: Gepstein. Circ Res 2002 & http://stemcells.nih.gov/info/media/DSC_1187.jpg Translation Derivation Regenerative Medicine Stem Cell Biology Differentiation Tissue morphogenesis Cellular therapies Propagation Scale UpScale Up Quantitative, systematicQuantitative, systematic approaches approaches Quality controlQuality control Stem cell-based therapy iPSCs Human somatic cells “Personalized medicine”

33. Disease Modeling using iPSCs Marchetto, M.C.N., Carromeu, C., Acab, A., Yu, D., Yeo, G. W., Mu, Y., Chen, G., Gage, F.H., and Muotri, A.R. (2010). A Model for Neural Development and Treatment of Rett Syndrome Using Human Induced Pluripotent Stem Cells. Cell, 143, 527-539. Lee, G., Papapetrou, E.P., Kim, H., Chambers, S.M., Tomishima, M.J., Fasano, C.A., Ganat, Y.M., Menon, J., Shimizu, F., Viale, A., Tabar, V., Sadelain, M., and Studer, L. (2009). Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402-406. Disease-specific iPSCs Disease-related differentiated cells